Project Summary The goal of this project is to develop a powerful platform that combines cutting-edge microscopy methods, image analysis algorithms, microfluidics, and macromolecular synthesis techniques to comprehensively characterize the dynamics of nucleic acids in well-controlled, time-varying biomimetic environments. The long-term vision is to characterize nucleic acid dynamics in the intracellular milieu, with a focus on large naked DNA, specifically motivated by recently engineered nucleic acid complexes for gene therapy and genomes of recently discovered macroviruses. The primary hindrance to understanding intracellular nucleic acid transport is the overwhelming complexity and diversity of macromolecules and networks that crowd cells. While many studies have explored macromolecular dynamics in crowded environments, experiments have largely been carried out either in: monodisperse, steady-state in vitro crowded systems, that fail to replicate the complex intracellular environment; or highly heterogeneous, dynamic in vivo systems, which offer incomplete understanding of the macromolecular components and properties responsible for observed transport. The proposed comprehensive experimental toolset will be designed to bridge this gap, and to directly couple single-macromolecule transport to (i) conformational dynamics, (ii) ensemble transport, and (iii) crowded environment properties. The vital first step towards applying this platform to cells is to design and utilize well-controlled in vitro cytoskeleton networks that can be precisely tuned over a wide parameter space to generate the rich DNA dynamics observed in vivo and to couple observed dynamics to tunable variables that include properties of both DNA and cytoskeleton. Environmental and DNA parameters will be varied and the resulting DNA and network dynamics will be measured using a platform consisting of (a) light-sheet microscopy with methods to probe single-molecule and ensemble DNA dynamics as well as environmental properties, (b) biomimetic cytoskeleton environments comprised of varying amounts of (i) actin and (ii) microtubules, (c) home-built microfluidic perfusion chambers for in situ modulation of polymerization states of (i) and (ii) in real-time, and (d) custom-engineered fluorescent-labeled DNA molecules of varying lengths and topologies. The current research will focus on optimizing and disseminating this robust platform, SLAMMTAP (Spatiotemporal Light-sheet Assisted Multiscale Macromolecular Transport Analysis Probe), and proving its utility and applicability to health-science researchers by identifying the key characteristics of both DNA (size, topology) and cytoskeleton environments (concentration, stiffness, crosslinking density, spatiotemporal heterogeneities) that lead to single-molecule transport, conformational dynamics and collective diffusion of DNA that mimic complex phenomena observed in vivo. Ultimately, in vitro and in vivo studies of DNA dynamics allowable by the techniques developed in this project will shed light on how viral genomes traverse the crowded cytoplasm and will help guide researchers to engineer DNA- or RNA-containing gene therapies with optimal efficacy.